ABSTRACT The p53 family of proteins regulate diverse physiological processes, from the cellular response to DNA damage to tissue homeostasis and development, through the ability to bind and respond to regulatory DNA sequences in the genome. All members of the family (p53, p63, and p73) possess pioneer factor activity, whereby they are capable of binding to specific DNA sequences in the context of compacted chromatin. Pioneer factors control de novo enhancer specification, a process required for cell lineage determination during development and appropriate regulation of stress and stimulus-dependent transcriptional programs. While pioneer factors are necessary, combinatorial activity of other transcription factors is critically important for enhancer activation. Our long-term goal is to dissect the mechanisms controlling p53 family-dependent enhancer specification, activity, and regulation. The objective of this grant is to define the activities of p63 (an epithelial-specific p53 family member) and co-operating transcription factors at transcriptional enhancers and during enhancer specification. The central hypothesis is that p63 uses its intrinsic pioneer factor activity to establish and regulate both DNA damage-specific and epithelial-specific enhancer networks. Our specific aims will test our central hypothesis by dissecting the mechanisms used by p63 to establish epithelial-specific enhancer networks and by investigating the mechanisms of by which p63 controls and specific p53-dependent DNA damage response. Collectively, the proposed research will impact our basic understanding of how this critical family of pioneer transcription factors function to regulate both common and cell-type specific enhancer networks. This is significant, as malfunction of p63 and p53 are directly involved in both cancer and severe developmental disorders, and cis-regulatory activity controls proper regulation of development and cellular homeostasis. The results and findings of this work can then be extended to model the activity of other pioneer transcription factors at both cell lineage and stress-dependent enhancers.